The objective of protein characterization during biopharmaceutical development and manufacturing is to decrease the time-to-result and improve data quality by eliminating the variability in manual protocols such as slab-gel electrophoresis, Western blotting, and HPLC. Ultimately these benefits translate to an accelerated time-to-market for new biologics.
Protein characterization is an integral step in biopharmaceutical development from the early stage of clone selection to final product characterization.
“Scientists are searching for screens and technologies that can be implemented early in development that are more predictive of performance during manufacturing to produce a high yield of product,” says Susan Harrison Uy, scientific content marketing manager at ProteinSimple, a business unit of BioTechne, “but running screens during early stages often involves analyzing low volumes of complex mixtures.”
The company’s Simple WesternTM size-based assays have sample requirements of just 3–5 μL, and a broad dynamic range of protein detection. The low sample requirements allows clones to be screened earlier than with traditional technologies, such as Western blots. Screening with Simple Western also provides confirmation of molecular weight, which is impossible with conventional immunoassay-based screens such as ELISA.
Protein characterization is an integral step in biopharmaceutical development.
“ELISA tells you if an analyte is present in a sample and quantifies it but doesn’t pick up changes in size. If an antibody can detect total target and there is a change in phosphorylation you can see that with Western blot but not ELISA,” Uy explains. Simple Western also detects host-cell proteins.
Characterizing charge heterogeneity of a biotherapeutic molecule is critical for product safety, purity, and potency, Uy adds. “Such factors as post-translational modifications often change the isoelectric point of the molecule and can affect product safety and potency. Simple Western charge assays simplify charge characterization in complex cultures, giving you quantitative, reproducible results.”
ProteinSimple also offers an HCP assay that automates preparation of a standard curve and occurs entirely within a microfluidic cartridge with their Ella platform. The company reports the ability to set up 72 samples in less than 15 minutes, with results in one hour, representing a significant time and labor savings over ELISA.
Special molecules, special methods
Last year KBI Biopharma acquired Alliance Protein Laboratories (APL), a biopharmaceutical analytical services company specializing in biophysical characterization of therapeutic proteins, nucleic acids, and vaccines. Alliance is known for its expertise in analytical ultracentrifugation, a method for characterizing proteins’ physical stability, association state, molecular weight, and state of aggregation. The company was one of the first to employ biophysical methods that demonstrate the similarity of higher-order structural states between biosimilar proteins and generic peptides to the innovator products.
“We’ve been performing biophysical analysis of biosimilars since 2003 and have already worked on nearly all the different biosimilars currently under development,” says John Philo, director of biophysical chemistry at APL.
APL has also worked with generic peptide drugs and bispecific antibodies. Although it does not work with cytotoxic drugs it can support antibody-drug conjugates through characterization of the antibody component.
The company’s specialty is sedimentation velocity analytical ultracentrifugation (SV-AUC), which physically separates and quantifies the desired product from aggregates by virtue of sedimentation rate (heavier species sediment more rapidly under centripetal forces).
SV-AUC measures stability by monitoring changes in aggregate content or aggregate types over time. It also detects changes in the conformation of the major component (the desired product) because such changes in higher-order structure alter the sedimentation rate.
Biocompare’s Proteomic Tools Search
Find, compare and review proteomic
tools from different suppliers Search
“However, since SV-AUC throughput is low, it isn’t used routinely to monitor stability samples,” Philo reports. “It is more commonly used to cross-validate other higher-throughput and less-costly stability assays such as size-exclusion chromatography.”
”For biosimilars or generic peptides, biophysical methods play a key role in demonstrating comparability of higher-order structure (secondary, tertiary, and quaternary structure). Ensuring that the biosimilar has the same solution conformation is critical for bioactivity (efficacy), and ensuring that it doesn’t contain more aggregates or sub-visible particles that could trigger unwanted immune reactions (immunogenicity) is critical for safety. The techniques used most commonly for biosimilarity studies are SV-AUC, circular dichroism (CD), and differential scanning calorimetry (DSC).”
For new products or next-gen proteins biophysical analysis can be helpful in selecting the best candidate protein to predict stability and/or “developability. Biomanufacturers also apply biophysical analysis in discovery research to directly measure the interactions of candidate drugs with their intended targets, as an aid to measuring bioactivity/potency, or to help elucidate the mechanism of action.
Yet for characterization of complex biotherapeutics no single method or approach stands alone. “The interpretation of biophysical data is stronger and more useful when the biophysical data are combined with data from other analytical methods to characterize the purity and homogeneity of the protein, such as mass spectroscopy, HPLC, CE, and glycan analysis,” Philo adds.
Host cell proteins
Residual host cell proteins (HCPs) carried over from expression systems are a potential safety and regulatory risk for biopharmaceuticals. Their risks, for example immunogenic reactions, do not necessarily correlate with their quantities as even small traces of certain HCPs can be highly immunogenic.
Many HCPs have physio-chemical properties that cause them to co-purify with the drug protein. The complexity of the HCP challenge requires a holistic approach to achieve a maximum of specificity/coverage for the analytical tools used during process development and in QC testing.
Since HCPs are a fact of life in all biopharmaceuticals, their quantification and characterization at all stages of downstream purification is a top priority.
“Biopharmaceutical manufacturers are expected to demonstrate that their purification train removes HCPs to a certain threshold, and this specification is specific to the product,” explains Olaf Stamm, senior business development specialist, Charles River Laboratories (CRL). In contrast to viral-clearance studies, success is measured by analyzing samples from actual purification stages.
In the past, biomanufacturers used immunoassays that provided an aggregate number for the entire complex mixture of several hundred HCPs in a batch. Today manufactures look much more closely and undertake HCP risk assessments, which requires deep understanding of potential HCPs of concern and how purification ensures their removal.
CRL has combined proteomics, bioinformatics, protein synthesis, and immunology into a service, HCP-GAPexsm, which carries the tagline of “the first holistic approach to host cell protein assay development.”
Assay characteristics differ depending on the product’s lifecycle. Generic assays will serve development and early-phase clinical stages for either E. coli or CHO expression cells. These assays are presented as validated kits based on normally expected HCPs.
During Phase III and beyond, developers turn to validated, product- and process-specific HCP assays. CRL custom-designs these assays through HCP-GAPex.
“Our understanding of risks associated with individual HCPs is still quite limited,” Stamm says, “but emerging tools that focus on analytics, like mass spectrometry, and molecular biology, like CRISPR/Cas9, will ultimately lead to expression systems that are engineered to eliminate high risk HCPs on a genomic level.
Image: Note how the black data points (originator molecule) and red data points (biosimilar) overlap. Image courtesy of KBI Biopharma.